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Zonal Sedimentation Analysis on Sucrose Gradients
蔗糖密度梯度分区沉淀分析   

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Abstract

Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein hemagglutinin (HA). Here, I describe step by step a protocol used to evaluate the oligomeric state of recombinant HA trimers (Magadan et al., 2013).

Materials and Reagents

  1. Trimerized recombinant HA (recHA3) derived from influenza A/Puerto Rico/8/34 (PR8) virus (Magadan et al., 2013)
  2. Gel filtration protein standards [carbonic anhydrase (29 kDa), ovalbumin (43 kDa), conalbumin (75 kDa), aldolase (158 kDa), and ferritin (440 kDa)] (GE, catalog numbers: 28-4038-41 and 28-4038-42 )
  3. 4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
  4. NuPAGE Novex 4-12% Bis-Tris protein gels (Life Technologies, catalog number: NP0321PK2 )
  5. NuPAGE MES SDS running buffer (Life Technologies, catalog number: NP000202 )
  6. Ponceau S solution (Sigma-Aldrich, catalog number: P7170 )
  7. 5% acetic acid
  8. Blotting grade blocker nonfat dry milk (Bio-Rad Laboratories, catalog number: 170-6404XTU )
  9. 1x PBS (Life Technologies, catalog number: AM9624 )
  10. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  11. A home-made, conformation-independent mouse monoclonal antibody to denatured HA1 (clone CM-1)
  12. A rabbit polyclonal anti-mouse HRP-conjugated antibody (Dako, catalog number: P0260 )
  13. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34077 )
  14. High purity sucrose (Thermo Fisher Scientific, catalog number: NC0110097 )
  15. UltraPure 5 M NaCl (Life Technologies, catalog number: 24740-011 )
  16. UltraPure 0.5 M EDTA (pH 8.0) (Life Technologies, catalog number: 15575-020 )
  17. Sucrose gradient (see Recipes)

Equipment

  1. Refractometer (Bausch & Lomb Incorporated)
  2. 14 x 89 mm Ultra Clear tubes (Beckman Coulter, catalog number: 344059 )
  3. Pipettor
  4. 1.5 ml micro-centrifuge tubes
  5. An ultracentrifuge equipped with a SW41 rotor (Beckman Coulter)
  6. Nitrocellulose blotting membranes (0.45 µm pore size) (Life Technologies, catalog number: LC2000 )
  7. A chamber to run mini-gels [I routinely use the XCell SureLock Mini Cell electrophoresis system (Life Technologies, catalog number: EI0001 ).]
  8. A Mini Trans-Blot Cell (Bio-Rad Laboratories, catalog number: 170-3930 )
  9. Carestream Kodak BioMax XAR films (Sigma-Aldrich, catalog number: F5388 )
  10. Kodak X-OMAT 2000A processor or equivalent
  11. A cassette for autoradiography

Software

  1. ImageJ software (http://imagej.nih.gov/ij/)

Procedure

  1. Zonal sedimentation on a sucrose gradient
    1. Simultaneously load 6 µg of recHA3 and 60 µg of each protein standard by pipetting them (use a 10 µl tip connected to a pipettor) just below the top of a 5-25% sucrose gradient containing a 60% sucrose cushion.
    2. Ultracentrifuge the sucrose gradient for 16 h at 35,000 rpm, 4 °C.
    3. Place the tube containing the sucrose gradient in a tightly fitted rack to avoid any undesired movement.
    4. Carefully place a 1 ml tip connected to a pipettor just below the top of the sucrose gradient.
    5. Manually collect fractions of 250 µl by slow pipetting.
    6. Transfer fractions to new 1.5 ml micro-centrifuge tubes.
    7. Repeat steps A4 and A6 until the sucrose gradient is completely fractionated.
    8. Combine 15 µl of each fraction with 5 µl of 4x LDS sample buffer.
    9. Boil samples for 5 min.

  2. SDS-PAGE and Western blotting
    1. Load 15 µl of every sample onto protein mini-gels.
    2. Run for ~1 h at constant 50 mA/gel.
    3. Transfer proteins to nitrocellulose membranes for ~1 h at constant 300 mA.
    4. Stain membranes with 10 ml 0.1% Ponceau S solution in 5% acetic acid for 5 min. Rinse membranes with water. At this point, it is possible to see the protein standards on the stained membranes.
    5. Scan or take a picture of the stained membranes.
    6. Block membranes with 10 ml 5% nonfat milk/1x PBS for 30 min at room temperature.
    7. Wash membranes with 10 ml 0.5% Tween-20/1x PBS for 10 min at room temperature with vigorous shaking.
    8. Incubate membranes with 10 ml neat mouse hybridoma supernatant containing the anti-HA antibody for at least 2 h at room temperature with slow rocking.
    9. Wash membranes 3 times with 10 ml 0.5% Tween-20/1x PBS for 10 min at room temperature with vigorous shaking.
    10. Incubate membranes with 10 ml anti-mouse HRP-conjugated antibody diluted 1:3,000 in 5% nonfat milk/1x PBS for 1 h at room temperature with slow rocking.
    11. Wash membranes 3 times with 10 ml 0.5% Tween-20/1x PBS for 10 min at room temperature with vigorous shaking.
    12. Incubate membranes with 5 ml SuperSignal West Pico chemiluminescent substrate for 5 min at room temperature with slow rocking.
    13. Expose films to membranes at room temperature.
    14. Develop films using a Kodak X-OMAT 2000A processor or equivalent.
    15. Please refer to Figure 5 on our prior publication (Magadan et al., 2013) for representative results and conclusions.

  3. Calculation of the recHA3 molecular size
    1. Measure the intensity of each protein standard band on stained membranes with Ponceau S using the Image J software. Then, plot the distribution of the protein standards on the different sucrose gradient fractions.
    2. Repeat step C1 measuring the distribution of recHA3 obtained by Western blotting.
    3. By comparing both plots, it is evident that recHA3 (~200 kDa) sediments as discrete peaks immediately following the fractions containing aldolase (158 kDa).

Recipes

  1. Sucrose gradient
    Prepare a 60% sucrose stock by dissolving high purity sucrose in 50 mM Tris-HCl (pH 7.5) and 100 mM NaCl
    Measure the refractive index (RI) of the 60% sucrose solution using a refractometer (RI=1.4418 at room temperature)
    Make 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, and 5% sucrose solutions using the 60% sucrose stock solution described above in this recipe
    Pour a 2 ml 60% sucrose cushion in a 14 x 89 mm tube and then 1 ml of the following sucrose solutions by careful pipetting: 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, and 5%
    Equilibrate the sucrose gradient for at least 1 h at room temperature

Acknowledgments

This protocol has been adapted from a previously published paper (Magadan et al., 2013). This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

References

  1. Magadan, J. G., Khurana, S., Das, S. R., Frank, G. M., Stevens, J., Golding, H., Bennink, J. R. and Yewdell, J. W. (2013). Influenza A virus hemagglutinin trimerization completes monomer folding and antigenicity. J Virol 87(17): 9742-9753.

简介

对蔗糖梯度的区域沉降分析允许通过在非变性条件下以恒定速度离心来估计单个蛋白质或蛋白质复合物的分子大小。 该方法特别适用于如甲型流感病毒(IAV)蛋白血凝素(HA)的球状蛋白。 在这里,我逐步描述用于评估重组HA三聚体的寡聚状态的方案(Magadan等人,2013)。

材料和试剂

  1. 来自流感A /波多黎各/8/34(PR8)病毒(Magadan等人,2013)的三聚体重组HA(recHA 3)
  2. 凝胶过滤蛋白标准[碳酸酐酶(29kDa),卵白蛋白(43kDa),伴白蛋白(75kDa),醛缩酶(158kDa)和铁蛋白(440kDa)](GE,目录号:28-4038-41和28 -4038-42)
  3. 4x NuPAGE LDS样品缓冲液(Life Technologies,目录号:NP0007)
  4. NuPAGE Novex 4-12%Bis-Tris蛋白凝胶(Life Technologies,目录号:NP0321PK2)
  5. NuPAGE MES SDS运行缓冲液(Life Technologies,目录号:NP000202)
  6. Ponceau S溶液(Sigma-Aldrich,目录号:P7170)
  7. 5%乙酸
  8. 印迹级阻断剂脱脂奶粉(Bio-Rad Laboratories,目录号:170-6404XTU)
  9. 1x PBS(Life Technologies,目录号:AM9624)
  10. Tween-20(Sigma-Aldrich,目录号:P1379)
  11. 对变性HA1(克隆CM-1)的自制的,构象独立的小鼠单克隆抗体
  12. 兔多克隆抗小鼠HRP缀合的抗体(Dako,目录号:P0260)
  13. SuperSignal West Pico Chemiluminescent Substrate(Thermo Fisher Scientific,目录号:34077)
  14. 高纯度蔗糖(Thermo Fisher Scientific,目录号:NC0110097)
  15. UltraPure 5M NaCl(Life Technologies,目录号:24740-011)
  16. UltraPure 0.5M EDTA(pH 8.0)(Life Technologies,目录号:15575-020)
  17. 蔗糖梯度(参见配方)

设备

  1. 折射计(Bausch& Lomb Incorporated)
  2. 14×89mm Ultra Clear管(Beckman Coulter,目录号:344059)
  3. Pipettor
  4. 1.5 ml微量离心管
  5. 装有SW41转子(Beckman Coulter)的超速离心机
  6. 硝酸纤维素印迹膜(0.45μm孔径)(Life Technologies,目录号:LC2000)
  7. 运行微型凝胶的小室[我常规使用XCell SureLock Mini Cell电泳系统(Life Technologies,目录号:EI0001)。]
  8. Mini Bio-Blot Cell(Bio-Rad Laboratories,目录号:170-3930)
  9. Carestream Kodak BioMax XAR膜(Sigma-Aldrich,目录号:F5388)
  10. 柯达X-OMAT 2000A处理器或等同设备
  11. 用于放射自显影的暗盒

软件

  1. ImageJ软件( http://imagej.nih.gov/ij/

程序

  1. 蔗糖梯度上的区带沉降
    1. 同时通过吸取它们(使用连接到移液器的10μl吸头)加载6μg的recHA 3和60μg的每种蛋白质标准品,恰好低于5-25%蔗糖梯度的顶部,其含有60 %蔗糖垫。
    2. 在4℃以35,000rpm超速离心蔗糖梯度16小时
    3. 将含有蔗糖梯度的试管置于紧密贴合的架子中,以避免任何不希望的运动
    4. 小心地将1毫升吸头连接到移液器正下方的蔗糖梯度的顶部。
    5. 通过慢速移液手动收集250μl的馏分
    6. 将馏分转移到新的1.5ml微量离心管中
    7. 重复步骤A4和A6,直到蔗糖梯度完全分馏
    8. 将每份15μl与5μl4x LDS样品缓冲液混合
    9. 煮沸样品5分钟。

  2. SDS-PAGE和Western印迹
    1. 加载15微升的每个样品到蛋白质小凝胶上
    2. 在恒定的50mA /凝胶下运行〜1小时
    3. 转移蛋白质到硝酸纤维素膜约1小时,在恒定的300毫安
    4. 用10%0.1%Ponceau S在5%乙酸中的溶液染色膜5分钟。 用水冲洗膜。 在这一点上,可以在染色的膜上看到蛋白质标准
    5. 扫描或拍摄染色膜的图片。
    6. 在室温下用10 ml 5%脱脂奶粉/1 x PBS封闭膜30分钟
    7. 在室温下用10ml 0.5%Tween-20/1×PBS洗涤膜10分钟,同时剧烈摇动
    8. 用含有抗HA抗体的10ml纯净小鼠杂交瘤上清液在室温下用缓慢摇动孵育膜至少2小时。
    9. 用10ml 0.5%Tween-20/1x PBS在室温下振荡洗涤膜3次,每次10分钟。
    10. 孵育膜与10ml抗小鼠HRP缀合的抗体稀释1:3,000在5%脱脂奶/1 x PBS在室温下1小时,缓慢摇摆。
    11. 用10ml 0.5%Tween-20/1x PBS在室温下振荡洗涤膜3次,每次10分钟。
    12. 孵育膜与5毫升SuperSignal West Pico化学发光底物在室温下摇动5分钟。
    13. 在室温下将膜暴露于膜
    14. 使用Kodak X-OMAT 2000A处理器或等同设备制作胶片。
    15. 请参阅我们之前的出版物(Magadan等人,2013年)上的图5,以获得有代表性的结果和结论。

  3. recHA 3分子大小的计算
    1. 使用Image J软件,用Ponceau S在染色的膜上测量每个蛋白质标准条带的强度。 然后,绘制蛋白质标准品在不同蔗糖梯度馏分上的分布
    2. 重复步骤C1,测量通过Western印迹获得的recHA亚基3的分布
    3. 通过比较两个图,显然recHA 3(约200kDa)作为离散峰在含有醛缩酶(158kDa)的级分后立即沉淀。

食谱

  1. 蔗糖梯度
    通过将高纯度蔗糖溶解在50mM Tris-HCl(pH 7.5)和100mM NaCl中制备60%蔗糖原料 使用折射计(室温下RI = 1.4418)测量60%蔗糖溶液的折射率(RI)。
    使用上述配方中的60%蔗糖储备溶液制备25%,22.5%,20%,17.5%,15%,12.5%,10%,7.5%和5%蔗糖溶液
    在14×89mm试管中倒入2ml 60%蔗糖垫,然后通过小心吸移将1ml以下蔗糖溶液:25%,22.5%,20%,17.5%,15%,12.5%,10% %,和5%
    在室温下平衡蔗糖梯度至少1小时

致谢

该协议已经改编自以前发表的论文(Magadan等人,2013年)。这项工作是由国家研究所的过敏和传染病,国家卫生研究院的校内研究支持。

参考文献

  1. Magadan,J.G.,Khurana,S.,Das,S.R.,Frank,G.M.,Stevens,J.,Golding,H.,Bennink,J.R.and Yewdell,J.W。(2013)。 甲型流感病毒血凝素三聚化完成单体折叠和抗原性。/em> 87(17):9742-9753。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Magadán, J. G. (2014). Zonal Sedimentation Analysis on Sucrose Gradients. Bio-protocol 4(8): e1100. DOI: 10.21769/BioProtoc.1100.
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